Cancer associated retinopathy (CAR) is a member of the paraneoplastic neuropathy family of autoimmune diseases and is characterized by the rapid progressive death of rod and cone photoreceptor cells, resulting in blindness. In CAR, the cancer is not in the eye, the visual cells degenerate with no detectable loss of other retinal neurons, no inflammatory response occurs, and the sera from a number of CAR patients have been shown to contain antibodies which react with recoverin, a soluble, calcium binding, retinal protein which acts by inhibiting the phosphorylation of rhodopsin by rhodopsin kinase. We propose to use a new in vitro model to test the hypothesis (Aim number 1) that antibodies against recoverin, can enter retinal neurons, react with recoverin and initiate a sequence of biochemical events which lead to the apoptotic death of rods, cones and recoverin positive bipolar cells. To accomplish this, the entry of immunoglobulins into retinal neurons and their reaction with recoverin will be characterized. Primary cell cultures of rat retinal neurons grown in a chemically defined medium, in combination with immunofluorescence microscopy, in situ hybridization, westerns, northerns, and a variety of biochemical and molecular biological techniques will be used (Aim number 2) to test the hypothesis that BAD, a proapoptotic Bcl-2-family member, is involved in the antibody-mediated death of retinal neurons along with specific mitochondria-associated apoptosis signal transduction proteins. We will also test the hypothesis (Aim number 3) that factors and/or reagents which stimulate the PI-3P and/or PKB signal pathways will also rescue recoverin positive retinal neurons from antibody-mediated cell death. Initial experiments with this system have already demonstrated the uptake of immunoglobulins by retinal neurons and the apoptotic death of rod photoreceptor cells in a time and dose-dependent manner following exposure to anti-recoverin IgG. In addition, intracellular calcium mobilization is shown to protect these retinal neurons from antibody-mediated apoptosis. It is anticipated that over the next five years our research will identify and characterize the mechanisms by which antibodies enter retinal neurons; the signal transduction pathways involved in the anti-recoverin antibody-mediated death of mammalian retinal neurons; and the cellular and molecular pathways by which these neurons can be rescued. The data from these studies may have direct applicability to the prevention of blindness in CAR as well as to the rescue of neurons in other paraneoplastic neuropathies.